High toxicity and specificity of the saponin 3-GlcA-28-AraRhaxyl-medicagenate, from Medicago truncatula seeds, for Sitophilus oryzae

<p>Abstract</p> <p>Background</p> <p>Because of the increasingly concern of consumers and public policy about problems for environment and for public health due to chemical pesticides, the search for molecules more safe is currently of great importance. Particularly, plants are able to fight the pathogens as insects, bacteria or fungi; so that plants could represent a valuable source of new molecules.</p> <p>Results</p> <p>It was observed that <it>Medicago truncatul</it>a seed flour displayed a strong toxic activity towards the adults of the rice weevil <it>Sitophilus oryzae</it> (Coleoptera), a major pest of stored cereals. The molecule responsible for toxicity was purified, by solvent extraction and HPLC, and identified as a saponin, namely 3-GlcA-28-AraRhaxyl-medicagenate. Saponins are detergents, and the CMC of this molecule was found to be 0.65 mg per mL. Neither the worm <it>Caenorhabditis elegans</it> nor the bacteria <it>E. coli</it> were found to be sensitive to this saponin, but growth of the yeast <it>Saccharomyces cerevisiae</it> was inhibited at concentrations higher than 100 μg per mL. The purified molecule is toxic for the adults of the rice weevils at concentrations down to 100 μg per g of food, but this does not apply to the others insects tested, including the coleopteran <it>Tribolium castaneum</it> and the Sf9 insect cultured cells.</p> <p>Conclusions</p> <p>This specificity for the weevil led us to investigate this saponin potential for pest control and to propose the hypothesis that this saponin has a specific mode of action, rather than acting <it>via</it> its non-specific detergent properties.</p

PA1b inhibitor binding to subunits c and e of the vacuolar ATPase reveals Its insecticidal mechanism

The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg-�ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. It also provides the first evidence for the position of subunit e within the comple

A folded and functional synthetic PA1b, an interlocked entomotoxic miniprotein

1-ACL (articles avec comité de lecture)PA1b (Pea Albumin 1, subunit b) is a hydrophobic, 37-amino acid niiniprotein isolated from pea seeds (Pivum sativum), crosslinked by three interlocked disulfide bridges, signature of the ICK (inhibitory cystine-knot) family. It acts as an entomotoxic factor against major insect pests in stored crops and vegetables, making it a promising bioinsecticide. Here we report an efficient and simple protocol for the production of large quantities of highly pure, biologically active synthetic PA1b. The features of PA1b oxidative refolding revealed the off-pathway products and competitive aggregation processes. The efficiency of the oxidative folding can be significantly improved by using hydrophobic alcoholic cosolvents and decreasing the temperature. The homogeneity of the synthetic oxidized PA1b was established by reversed-phase HPLC. The correct pairing of the three disulfide bridges, as well as the three-dimensional structure of synthetic PA1b was assessed by NMR. Synthetic PA1b binds to rnicrosomal proteins from Sitophilus oryzae with a Kd of 8 nM, a figure quite similar to that determined for PA1b extracted from its natural source. Moreover, the synthetic miniprotein was as potent as the extracted one towards the sensitive strains of weevils. Our findings will open the way to the production of PA1b analogues by chemical means to an in-depth understanding of the PA1b mechanism of action

High-affinity Nod Factor binding site form <i>Phaseolus vulgaris</i> cell suspension cultures

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Biological activity and binding site characteristics of the PA1b entomotoxin on insects from different orders

1-ACL (articles avec comité de lecture)The aim of this work was to investigate both the biological activity of an entomotoxin, the pea albumin 1b (PA1b), and the presence or absence of its binding site within an array of insect species. The data obtained showed that insect sensitivity was not related to its taxonomic position. Moreover, PA1b was not toxic to several tested microorganisms. However, the binding site was found to be conserved among very different insects, displaying similar thermodynamic constants regardless of the in vivo species sensitivity. The binding site alone was, therefore, not sufficient for toxicity. One exception was the pea weevil, Bruchus pisorum, which was the only tested species without any detectable binding activity. These findings indicate that the binding site probably has an important endogenous function in insects and that adaptation to pea seeds resulted in the elimination of the toxin binding activity in two independent insect lineages. Other mechanisms are likely to interact with the toxin effects, although they are still largely unknown, but there is no evidence of any specific degradation of PA1b in the midgut of insects insensitive to the toxin, such as Drosophila melanogaster or Mamestra brassicae

Toxicity, binding and internalization of the pea-A1b entomotoxin in Sf9 cells

1-ACL (articles avec comité de lecture)PA1b (Pea Albumin 1b) is a peptide toxin lethal for certain insects. This paper shows that the cultured insect cells Sf9 are sensitive to the toxin and display a high-affinity binding site for PA1b. Mammalian cells are not sensitive and no binding activity was detected. Signs of apoptosis of the Sf9 cells were observed in response to the toxin. The use of this cellular model also demonstrated that PA1b was internalized in the cells, via the binding site, raising the new question of the role of this toxin within the cell, and of the mechanisms leading to cell death

PA1b, a small knotted albumin from garden pea, protects transgenic rice against its major post-harvest insect pest Sitophilus oryzae

1-ACL (articles avec comité de lecture